Plant Physiology Preview. Published on May 27, 2011, as DOI:10.1104/pp.111.180042
Running head:
2b-CAT3 interaction induces necrosis
Corresponding Author:
Chikara Masuta
Kita9 Nishi9, Kita-ku, Sapporo 060-8589, JAPAN
Phone: +81-11-706-2807
Fax: +81-11-706-2483
E-mail: [email protected]
Journal Research Areas: Plants Interacting with Other Organisms
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Copyright 2011 by the American Society of Plant Biologists
Virus-induced necrosis is a consequence of direct protein–protein interaction
between a viral RNA silencing suppressor and a host catalase
Jun-ichi Inaba, Bo Min Kim, Hanako Shimura, Chikara Masuta*
Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8389, Japan
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Footnotes:
*Corresponding author; e-mail [email protected]; fax +81-11-706-2483
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ABSTRACT
Many plant host factors are known to interact with viral proteins during
pathogenesis, but how a plant virus induces a specific disease symptom still needs
further research. A lily strain of Cucumber mosaic virus (CMV-HL) can induce discrete
necrotic spots on infected Arabidopsis plants; other CMV stains can induce similar
spots, but they are not as distinct as those induced by CMV-HL. The CMV 2b protein
(2b), a known RNA silencing suppressor (RSS), is involved in viral movement and in
symptom induction. Using in situ proximity ligation assay (PLA) immunostaining and
the protoplast assays, we report here that CMV 2b interacts directly with catalase
(CAT3) in infected tissues, a key enzyme in the breakdown of toxic H2O2. Interestingly,
CAT3, normally localized in the cytoplasm (glyoxysome), was recruited to the nucleus
by an interaction between 2b and CAT3. Although overexpression of CAT3 in
transgenic plants decreased the accumulation of CMV and delayed viral symptom
development to some extent, 2b seems to neutralize the cellular catalase contributing to
the host defense response, thus favoring viral infection. Our results thus provide
evidence that, in addition to altering the type of symptom via disturbing miRNA
pathways, 2b can directly bind to a host factor that is important in scavenging cellular
H2O2 and thus interfere specifically with that host factor, leading to the induction of a
specific necrosis.
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Introduction
Cucumber mosaic virus (CMV) belongs to the genus Cucumovirus and contains
tripartite, single-stranded sense RNAs designated RNAs 1–3 in decreasing order of
molecular weight. RNAs 1 and 2 encode replicase proteins 1a and 2a, respectively, and
RNA3 encodes the movement protein 3a. A subgenomic RNA, RNA4 encodes the coat
protein (CP). The 2b protein (2b) is encoded in a subgenomic RNA, RNA4A, which has
a 3′-proximal ORF in RNA2 (Ding et al., 1994).
The CMV 2b protein is involved in local and systemic movement of CMV and
inhibits host defense through RNA silencing (Ding et al., 1995; Ji and Ding, 2001; Shi
et al., 2003; Soards et al., 2002) and also acts as a symptom determinant, which is
apparently associated with its RNA silencing suppressor (RSS) activity (Brigneti et al.,
1998; Lucy et al., 2000). The 2b protein contains two nuclear localization signals
(NLSs) in its N-terminal (Lucy et al., 2000; Goto et al., 2007) and has been confirmed
to be actually localized in the nucleus in infected tissues; the NLSs of the 2b protein are
required for viral symptom induction and RSS activity (Lewsey et al., 2009; Lucy et al.,
2000). Recently, 2b was found to bind to Argonaute 1 (AGO1), necessary for the plant
antiviral machinery, and to Argonaute 4 (AGO4), necessary for the DNA methylation
process that controls heterochromatin formation and transcription (Zhang et al., 2006;
Gonzalez et al., 2010). In addition, 2b was also reported to directly bind to small
interfering RNAs (siRNAs) (Goto et al., 2007; Kanazawa et al., 2010).
Hydrogen peroxide (H2O2), one of the reactive oxygen species (ROS), plays an
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important role in many physiological phenomena such as senescence, photorespiration
and photosynthesis, growth and development and resistance responses against
pathogens (Peng et al., 2005; Noctor and Foyer, 1998; Foreman and Tang, 2003). In
addition, H2O2 is
generated as a primary messenger to induce hypersensitive
cell death in the plant host. Catalase, which breaks down H2O2 and functions as a
cellular sink for H2O2, is an enzyme found in nearly all living organisms. In Arabidopsis
thaliana, the catalase gene family is composed of three members, CAT1, CAT2 and
CAT3 (Frugoli et al., 1996). CAT2 and CAT3 are localized in the peroxisome and are
the major H2O2 scavengers that control the ROS homeostasis in plants (Du et al., 2008).
The mutants, cat2 and cat3 typically display patches of chlorosis and necrotic lesions
(Contento and Bassham, 2010).
Although the lily strain of CMV (CMV-HL) can induce a necrosis on many
Arabidopsis ecotypes such as Col-0 and Ler, we chose Col-0 because many mutant lines
are available for future study. In Arabidopsis Col-0, CMV induces a distinct necrosis,
which is associated with the H2O2 that is generated after infection. In this paper, we
investigated a direct link between the CMV-induced necrosis and the 2b-CAT3
interaction and found that cell death was indeed induced by 2b’s binding to CAT3. Here,
on the basis of a simple and specific protein–protein interaction between 2b and CAT3,
we can begin to explain the molecular mechanism underlying CMV-induced necrosis in
Arabidopsis.
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RESULTS
Leaf necrosis in CMV-HL-infected Arabidopsis plants
CMV can systemically infect Arabidopsis and causes a variety of symptoms
depending on the particular combination of viral strain and Arabidopsis ecotype. After
the Col-0 plants were inoculated with CMV Y strain (CMV-Y), they developed systemic
mosaic symptoms 10 days after inoculation, and at 3 weeks after inoculation, necrotic
lesions were sometimes observed on the upper leaves. When Col-0 plants were
inoculated with the lily strain of CMV (CMV-HL), the virus was initially localized
within the inoculated leaves and spread very slowly to the upper leaves compared to
CMV-Y, but it induced more severe necrosis on the upper leaves than CMV-Y did (Fig.
1A). To identify the viral factor(s) for the necrosis induction, we created
pseudorecombinants between CMV-Y and CMV-HL. The three genomic RNAs of
CMV-Y and CMV-HL were designated Y1 to Y3 and H1 to H3, respectively. Among
the different pseudorecombinants between CMV-Y and CMV-HL, Y1H2Y3 induced
clear necrosis on the inoculated plants, suggesting that RNA2 is important for the
symptom. Instead of the original CMV-HL, we chose Y1H2Y3 for further analysis of
the observed necrosis, because unlike CMV-HL, it could systemically move in Col-0 as
fast as CMV-Y and induce very distinct necrosis on the upper leaves (Fig. 1 and data not
shown).
Y1H2Y3 induced distinct necrosis on the upper leaves at 10 dpi, while CMV-Y did
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not at this stage (Fig. 1B). The necrotic spots induced by Y1H2Y3 infection were found
to be associated with an increase in H2O2 production, as measured by oxidation of
H2DCFDA (Fig. 1C). Because H2O2 acts as an inducer of defense genes such as
pathogenesis-related protein (PR) genes, we analyzed the expression of some PR genes.
As a result, levels of PR1 and PR5 were enhanced, implying that the necrosis might be a
defense response (Supplemental Fig. S1). In tissue printing experiments, both CMV-Y
and Y1H2Y3 were distributed throughout the upper leaves, but the virus was not
detected in the centers of the coalescing necrotic spots (Fig. 1D). Because we found that
RNA2 of CMV-HL (H2) was important for the necrosis, we then asked whether the
RNA2-encoded proteins (2a and/or 2b) are involved in the necrosis. We focused on 2b
because it has been suggested that 2b is implicated in CMV symptoms including
necrosis (Du et al., 2008).
HL2b interacts with CAT3 in the yeast two-hybrid assay
To analyze how CMV 2b is involved in the necrosis induction, we started with the
isolation of host factors that interact with CMV 2b. We screened an A. thaliana cDNA
library for the yeast two-hybrid assay in the GAL4 activation domain (AD) vector
pGADT7 using the GAL4 binding domain (BD) bait vector that expresses the HL 2b
in-frame fusion with the BD (pGBKT7-HL2b). Although CMV 2bs had been reported
to act as a transcriptional activator in yeast (Ham et al., 1999; Sueda et al., 2010),
fortunately HL2b did not have this function. We screened about 5.0 × 106 independent
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yeast transformants, and eventually obtained eight candidate clones. One of the clones
that had strong β-galactosidase activity in the two-hybrid assay was selected for
sequencing and found to be the catalase3 gene (CAT3, AT1G20620). Because the
CAT3 gene isolated from the pGADT7 library was not complete, to verify the
interaction between HL2b and CAT3, we then cloned the full-length ORF of the CAT3
gene and re-examined it by the yeast two-hybrid assay; we here included two other
Arabidopsis catalase genes (CAT1 and CAT2). Each of the catalase genes (CAT1, CAT2
and CAT3) was cloned into the pGADT7 vector to create pGADT7-CAT1,
pGADT7-CAT2 and pGADT7-CAT3, respectively. Each construct was co-transferred
with pGBKT7-HL2b into Saccharomyces cerevisiae strain AH109. The results revealed
that HL2b indeed interacted with CAT3 but did not with the other two catalases (CAT1
and CAT2) (Fig. 2). To determine which domain of 2b is important for the interaction
with CAT3, we created several HL2b deletion mutants that lack the C-terminal amino
acids. When the C-terminal amino acids were deleted by 40 amino acid residues
creating HL2b∆40, the mutant 2b then lost the ability to bind to CAT3 in the two-hybrid
assay, and the virus (C2-H1) containing HL2b∆40 (C2-H1-HL2b∆40) also lost the
ability to induce necrosis, indicating a link between the binding of 2b to CAT3 and
necrotic induction (Fig. 3). C2-H1 is a CMV-Y-based virus vector in which a multiple
cloning site replaces the 2b gene (Matsuo et al., 2007). Therefore, the C-terminal region
of HL2b is important for the interaction between 2b and CAT3.
Catalase activity and necrosis in CMV-infected plants and transgenic plants
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expressing 2b
Because we had an idea that the interaction between 2b and CAT3 could induce the
necrosis, we then measured catalase activity in virus-infected tissues. CAT2 and CAT3
are highly expressed in leaves and thus account for most of the catalase activity. Both
cat2- and cat3-knockout plants displayed patches of necrotic lesions, suggesting that
reduction in either CAT2 or CAT3 causes necrosis in leaves (Contento and Bassham,
2010). In fact, catalase activity was significantly decreased by 10 dpi in the
Y1H2Y3-infected leaves when compared to the mock- or CMV-Y-inoculated control
plants (Fig. 4A). To investigate whether the mRNA levels of the Arabidopsis catalase
genes (CAT1-CAT3) were decreased in CMV infection, we measured those by real-time
RT-PCRs and found that there was no difference in any catalase genes at the mRNA
level (Fig. 4B). Western blots using CAT3-specific antibodies revealed that CAT3 was
not also changed at the protein level (Fig. 4C). To examine whether 2b is the
determinant for the necrosis induction by Y1H2Y3 in Col-0 leaves, we then generated
transgenic Arabidopsis plants that express the HL2b gene (Col-HL2b) (Fig. 5). In those
transgenic lines, we confirmed the accumulation of 2b by Western blot analyses using
anti-2b antibodies (Fig. 5B). Three transgenic lines developed necrosis on leaves at the
seedling stage in association with H2O2 formation, just as observed for CMV-infected
leaves (Fig. 5C). These results suggest that the expression of 2b alone causes necrosis
by generating H2O2 in Col-0 leaves, which is analogous to that observed in
CMV-infected Arabidopsis. We also measured catalase activities in the Col-HL2b plants
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and found that catalase activities in all the lines were indeed reduced below 50% of the
catalase activity of non-transgenic plants (Fig. 5D). We therefore considered that 2b
expression caused a decrease in catalase activity in the transgenic cells, which likely
caused H2O2 generation leading to necrosis.
HL2b interacts with CAT3 in Col-0 cells and alters the cellular location of CAT3
To confirm further the results of the two-hybrid assay and to determine whether
HL2b associates with CAT3 in Col-0 cells, we performed the Duolink in situ proximity
ligation assay (PLA), a method to detect proteins using two primary antibodies for
visualization and quantification of specific protein–protein interactions in situ
(Soderberg et al., 2008). According to the assay principle, only when two target proteins
are close to each other, a fluorescent signal will be detected. Col-0 protoplasts were
transfected with the plasmid constructs expressing HL2b, CAT3 with a FLAG-tag
(CAT3-FLAG) and GFP (control). In the cells transfected with HL2b+CAT3-FLAG,
strong fluorescent signals were localized in the nuclei (Fig. 6A), whereas no signal was
detected in the cells transfected with either of the plasmids alone, indicating that the
2b-CAT interaction was in the nuclei. Compared to the HL2b–CAT3 interaction, we
observed relatively weaker signals between Y2b and CAT3, indicating that Y2b binds
more weakly than HL2b to CAT3 (Supplemental Fig. S2). This is consistent with the
observation that CMV-Y did not induce necrosis on Arabidopsis as strongly as CMV-HL
did (Fig. 1). Subcellular localization of 2b and CAT3 was analyzed by the single
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recognition method in PLA using one primary antibody. We investigated whether the
interaction between 2b and CAT3 alters the original localization of 2b or CAT3. In the
protoplasts transfected with CAT3-FLAG alone, CAT3 signals visualized by anti-FLAG
antibodies diffused through the cytoplasm (Fig. 6B). When protoplasts were transfected
with HL2b+CAT3-FLAG, the CAT3 signals were not detected in the cytoplasm but
found in the nucleus, where 2b originally is localized (Fig. 6B and 6C), suggesting that
CAT3 was translocated into the nucleus with 2b. We also obtained similar results using
Y2b instead of HL2b, suggesting that the 2b proteins generally interact with CAT3 in
vivo although they differ in their intensity of interaction (Supplemental Fig. S2).
CAT3 overexpression enhanced tolerance to CMV infection
Because we found that a viral protein (2b) interacted with a host protein (CAT3) in
vivo, we then investigated whether overexpression of CAT3 affects not only CMV 2b
but also CMV infection (e.g., symptom appearance) in the transgenic plants. To obtain
Arabidopsis plants that overexpress the CAT3 gene, we transformed Col-0 plants using
the Ti-plasmid vector in which the CAT3 gene was inserted under the control of the 35S
promoter. We finally selected three transgenic lines (4, 6 and 7), which grew normally,
for inoculation experiments (Supplemental Fig. S3). By Western blot analyses, we
confirmed enhanced levels of CAT3 in those transgenic plants. In agreement with the
CAT3 levels, the catalase activities also increased in all three lines (Supplemental Fig.
S3). When those transgenic lines were inoculated with Y1H2Y3, at 10 dpi, none of the
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transgenic plants had developed necrosis, while non-transgenic plants had distinct
necrosis associated with H2O2 generation (Fig. 7A and 7B). In addition, necrosis was
delayed significantly according to a statistical analysis compared to the non-transgenic
plants, although all plants eventually developed necrosis, suggesting that elevated levels
of CAT3 only delayed the onset of necrosis (Fig. 7C).
CAT3 expression compromises cell death induced by 2b in protoplasts
To determine at the cellular level whether HL2b expression causes cell death, we
used the Arabidopsis protoplast system, which we had previously developed to analyze
the HR-like cell death induced by TuMV (Kim et al., 2010). When protoplasts were
transfected with the Ti-plasmid expressing HL2b, the percentage of cell death was
significantly increased. However, addition of the Ti-plasmid expressing CAT3 negated
the cell death induced by HL2b (Fig. 8A). Coexpression of HL2b and CAT3 led to a
decrease in catalase activity (Fig. 8B). These results thus suggest that HL2b can induce
cell death by binding to CAT3 and impairing the catalase activity and that the
HL2b-CAT3 interaction partially compromised the H2O2 scavenging by CAT3.
Effect of CAT3 on the silencing suppressor activity of 2b and CMV accumulation
levels in protoplasts
Fig. 9A shows that enhanced levels of CAT3 enabled the transgenic lines to suppress
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CMV multiplication at least until 7 dpi. However, at 14 dpi, the levels of CMV reached
those of the non-transgenic control plants for all three lines. These results indicate that
overexpression of CAT3 could confer partial tolerance at an initial stage of viral
infection. We hypothesized that CAT3 may inactivate the RNA silencing suppressor
(RSS) activity of 2b by binding with 2b, thus inhibiting CMV multiplication. To
investigate the effect of CAT3 on the RSS activity of 2b, we used a protoplast assay that
we have previously developed to measure viral RSS activities on a cellular level
(Shimura et al., 2008). As shown in Fig. 9B, CAT3 expression actually decreased the
RSS activity of 2b by 30–40%, but it did not affect another virus RSS, P19. On the
contrary, CMV infection did not downregulate CAT3; when non-transgenic plants were
inoculated with CMV, viral infection did not affect CAT3 either at the mRNA or the
protein level (Fig. 4).
The binding of 2b to CAT3 is important for cell death but the translocation of
CAT3 to the nucleus is not
Finally, to answer the question of which is important for the 2b-induced cell death,
the binding of 2b to CAT3 or the subsequent translocation of CAT3 to the nucleus, we
created a mutant HL2b (HL2b∆NLS) that lacks its nuclear localization signals (NLS1
and NLS2) (Fig. 10A). We then conducted the protoplast assays to observe cell death
and the localization of CAT3 and HL2b∆NLS. In situ PLA assays revealed that neither
HL2b∆NLS nor CAT3 was localized in the nucleus, but the two proteins bound to each
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other (Fig. 10B to 10D). As shown in Fig. 10E, HL2b∆NLS could still induce cell death
as efficiently as the authentic HL2b. These results thus indicate that the binding of 2b to
CAT3 is per se important to inhibit catalase activity.
DISCUSSION
Direct interactions between a viral and host factor in symptom induction
Many attempts to find host factors necessary for viral replication and movement,
which actually interact with viral proteins, have been made to elucidate the molecular
mechanisms for viral pathogenicity. For example, as reviewed by Ishibashi et al. (2010),
more than 10 host factors for multiplication of tobamoviruses have been identified
already. Those factors certainly play a role in viral pathogenicity as well as viral
multiplication, apparently serving as a host range or symptom determinant. There are
also many reports that describe symptom modifications as an indirect consequence of
the disturbance of the cellular physiology of the plant, such as host factors that interact
with viral RNA silencing suppressors against the host RNA silencing machinery, which
is essential not only for defense against viruses but also plant development. For example,
the CMV 2b protein can interfere with RNA silencing by binding to the host AGO1
protein, leading to disturbance of miRNA-directed physiology in CMV-infected plants
(Zhang et al., 2006). However, there are only a few reports that merely link
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protein–protein interactions between viral and host factors to distinct symptom
phenotypes as a direct result of a viral protein interfering with the intrinsic function of a
specific host factor. In one such study, Zhu et al. (2005) previously demonstrated a
direct interaction between the P2 protein of Rice dwarf virus (RDV) and rice
ent-kaurene oxidases, which play a role in the biosynthesis of gibberellins; this
interaction led to a decrease in the hormones and stunting of the infected plants. More
interestingly, they also raised the possibility that the interaction may actually also
decrease phytoalexin biosynthesis and make the plants more competent for viral
replication, suggesting that the interaction apparently works in favor of the virus. We
here provide more evidence strongly supporting a viral protein as a primary target for
determining the type of viral symptom expression. It is quite reasonable to speculate
that the necrosis formation in CMV-infected leaves is due to the decreased levels of
CAT activity as a result of the 2b–CAT3 interaction.
Other catalases in many
natural hosts for CMV including N. benthamiana may also bind to 2b
because database search revealed that their amino acid sequences were well
conserved. In fact, in our preliminary experiments, the Potato virus X (PVX)
vector over-expressing the intact Y2b but not a truncated Y2b lacking the
C-terminal 40 amino acids could induce necrosis on N. benthamiana.
The 2b–CAT3 interaction serves as a necrosis determinant in favor of CMV
infection
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The interaction between RDV P2 and ent-kaurene oxidases, as we have described it,
appears to work in favor of the virus RDV. Now, an interesting question is for which
side does the 2b–CAT3 interaction work? Does it elevate or reduce host defense after
all? We discuss this “two sides of the same coin” question taking the following
observations into account: (1) 2b bound to CAT3 and changed the localization of CAT3
from the cytosol to the nucleus in Col-0 protoplasts; (2) the expression of CAT3
inhibited the silencing suppressor ability of the coexpressed 2b in N. benthamiana
protoplasts and the necrosis induced by 2b in Col-0 protoplasts; (3) the overexpression
of CAT3 reduced the accumulation of CMV and delayed the necrosis in transgenic
Col-0 plants. When these results are considered together, CAT3 seems to have the
potential to inhibit CMV infection but normally not to sufficiently function as a natural
defense protein. In fact, it is not even induced by CMV infection (Fig. 4), and thus
Arabidopsis does not seem to have developed it as a defense system right against CMV.
However, we will not presume to deny this CAT3’s potential because there is certainly
the case where a host factor binds to a viral RSS to inhibit the RSS activity. For
example, in Tobacco mosaic virus (TMV), a host factor TOM1 binds to the RSS (130k
protein) of TMV and recruits it on membrane, resulting in reducing the RSS activity
(Yamanaka et al., 2000).
The interaction between a host and a viral factor must be also discussed from the
obverse side of defense. Catalase activity can be compromised by the binding of 2b to
CAT3. We here demonstrated that the translocation of CAT3 to the nucleus was not
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essential. However, the CAT3 malfunction is likely to be irreversible after CAT3 is
forced into the nucleus and thus it may still contribute to the necrosis formation in the
long run. Although this necrosis was shown to be HR-like, it is induced directly by a
simple protein-protein interaction but not in a decent signal transduction for defense.
Part of host defense responses may have been activated because some PR genes were
elevated, but CMV was not restricted with the necrosis at all, suggesting that any
coordinated defense system did not operate against CMV in association with this
2b-mediated necrosis.
It is therefore conceivable that a viral protein may bind to a host factor and/or
further alter the localization of the host protein to specifically induce a viral symptom.
Considering that most CMV strains do not cause necrosis in Arabidopsis, the
necrosis-inducing CMVs such as CMV-HL may have developed a strategy to
interfere with the catalase-mediated defense to more efficiently multiply by
establishing the binding between 2b and catalase in the viral evolution. In
conclusion, our study here suggests that the HL2b–CAT3 interaction serves as a
necrosis determinant, rather than a potential defense mechanism, perhaps in favor of the
virus.
MATERIALS AND METHODS
Plant materials and growth conditions
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The ecotypes of Arabidopsis thaliana, Columbia (Col-0) and the transgenic plants
expressing HL2b or CAT3, were used in these experiments. Seeds were directly sown in
peat pellets and grown in MLR-350 growth chamber (SANYO, Tokyo, Japan) at 21 °C
with a 12 h photoperiod (150 µmol⋅m−2⋅s−1).
Plant transformation
The cDNAs of HL2b and CAT3 were inserted into the multiple cloning site of a plant
expression vector pBE2113 (Mitsuhara et al., 1996). Agrobacterium tumefaciens strain
EHA105 was first transformed with the vector construct by the freeze and thaw method
(Holster et al., 1978). Col-0 plants were then transformed with the pBE2113-HL2b or
-CAT3 by the vacuum infiltration method (Bechtold et al., 1993). Transformants were
selected on a solid medium containing 2% sucrose supplemented with 1/2 Murashige
and Skoog (MS) nutrients and kanamycin (50 μg/mL).
Viral inoculation and H2O2 detection
The plasmids containing the full-length cDNAs of RNAs of CMV-Y or CMV-HL
were transcribed in vitro. The leaves of 4-week-old plants of N. benthamiana were
dusted with carborundum and rub-inoculated with the in vitro-transcribed RNAs. For
Arabidopsis, 3-week-old plants were inoculated with the sap of an infected N.
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benthamiana leaf. Successful infection was confirmed by conventional ELISA (Kim et
al., 2010). Viral infection was confirmed using tissue prints and anti-CMV antibodies
essentially as described by Kaneko et al. (2004). H2O2 generation in leaf tissues was
detected using H2DCFDA (Kim et al., 2008).
Quantitative real-time RT-PCR for CMV RNAs
Total RNA was isolated using Trizol reagent (Invitrogen, Tokyo) as described
previously (Kim et al., 2008). For the real-time RT-PCR, 1 µg of total RNA was used as
a template for cDNA synthesis. First strand of cDNA was synthesized by the Takara
RNA PCR Kit (Takara, Ohtsu, Japan) with a random primer. Real-time RT-PCR was
carried out using 1 µL of the RT reaction mixture and SYBR green mixture (Takara,
Ohstu, Japan) in the DNA Engine Opticon 2 System (MJ Research, Waltham, MA,
USA). A specific primer pair for the 3′-end sequence of CMV RNA3 (352 bp) was used
to measure CMV RNAs (Yamaguchi et al., 2005). For the amplification of the
Arabidopsis CAT genes, the following primer pairs were used: At-CAT1-5
(5′-ATCTGCTTGAGAAACTCGCTAACT-3′)
and
(5′-ATGACAGTTGAGAAACGAACGATA-3′)
for
(5′-GGATGGTTCAGGTGTCAATACATA-3′)
(5′-GATAAAGAGCTTCCATTCAGGGTA-3′)
GGAATTCGAACAAAGTGCGT-3′)
and
At-CAT1-3
CAT1,
and
for
CAT2,
At-CAT2-5
At-CAT2-3
At-CAT3-5
At-CAT3-3
(5′
(5′-
－
AAGGATCGATCAGCCTGAGA-3′) for CAT3. For all the Arabidopsis genes analyzed
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by real-time PCR, the β-tubulin sequence was amplified as an internal control using a
primer
pair
of
At-tub-5-200
(5′-GAGGGAGCCATTGACAACATCTT-3′)
and
At-tub-3-200 (5′-GCGAACAGTTCACAGCTATGTTCA-3′).
Western blot analysis
Total proteins from Col-0, Col-HL2b and Col-CAT were extracted as described by
Masuta et al. (1995). The samples were first boiled for 5 min, separated on a 15% SDS
page and blotted to a PVDF membrane (Millipore). The blots were probed with
antibodies against CMV 2b or CAT3. Proteins were visualized using anti-mouse
secondary antibodies conjugated to alkaline phosphatase, followed by treatment with
NBT/BCIP as described previously (Sato et al., 2003).
Detection of catalase activities
Total protein of Arabidopsis seedlings was extracted from 0.1 g tissues by grounding
to a fine powder in liquid nitrogen and homogenized in 0.4 mL of the extraction buffer
(50 mM phosphate buffer, pH 7.0 and 1% Triton X-100). After centrifugation at 12000
× g for 10 min at 4 °C, the proteins in the supernatant were quantified by a protein
assay kit (Bio-Rad). In the catalase activities assay, 50 μg of total protein was added to
0.9 mL of the buffered-substrate (50 mM phosphate buffer, pH 7.0 and 20 mM H2O2).
The degradation of H2O2 was determined by a decline in absorbance at 240 nm.
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Immunostaining by in situ PLA
Protoplasts were isolated from 4-week-old seedlings of Col-0 and transfected with
the
plasmids
(GFP,
HL2b,
Y2b,
CAT3-FLAG,
HL2b+CAT3-FLAG
or
Y2b+CAT3-FLAG) using PEG-calcium-mediated transformation (Kim et al., 2010).
The cDNA clones of HL2b, Y2b and CAT3-FLAG were inserted into the cloning site in
pBE2113 (Mitsuhara et al., 1996), and the control GFP gene was also cloned in pE2113.
These protoplasts were fixed with 2% paraformaldehyde on L-polylysine-coated slides
(Yoo et al., 2007; Tariq et al., 2003) 24 h after transfection. After rehydration in PBS,
the fixed protoplasts on the slides were blocked in 2% BSA in PBS (30 min, room
temperature) and incubated one hour at room temperature in 0.5 % BSA in PBS
containing primary antibodies. A protein–protein interaction was detected using
antibodies against CMV 2b or FLAG with Duolink in situ PLA probes (OLINK
BIOSIENCE). The images were observed with a fluorescence microscope (Leica DMI
6000B). Red signals (Texas Red) were visualized with excitation at 543 nm (emission:
585 to 615 nm), and blue signals (UV) were visualized with excitation at 365 nm
(emission: 420 to 470 nm).
Observation of cell death in protoplasts
Cell death of protoplasts was assessed with Evans blue (0.04%, w/v) staining 20 h
22
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Copyright © 2011 American Society of Plant Biologists. All rights reserved.
after transfection as described previously (Kim et al., 2010). Our average transfection
efficiency was approximately 70%. We counted the number of dead cells in several
fields at 400×. Protoplasts were exposed to the dye for 5 min, and then observed with
light microscopy (Olympus BX51; Olympus, Tokyo).
Protoplast assay for RSS activity
Protoplasts were prepared from leaves of N. benthamiana and used to analyze RSS
activity as essentially described by Shimura et al. (2008). For this assay, we used the
Dual-Luciferase Reporter Assay System (Promega Corp., Madison, WI, USA) using
firefly luciferase (Fluc) and Renilla luciferase (Rluc). As a silencing inducer,
double-stranded (ds) RNA of the Fluc gene (dsFluc) was used. RNA silencing in
protoplasts was induced by co-transfection of the reporter genes and dsFluc with or
without the plasmids containing a viral suppressor gene, CMV-HL2b or P19 of Tomato
bushy stunt virus (TBSV). The protoplasts were then harvested 24 h after transfection
and analyzed for the luciferase activity.
Supplemental Data
Supplemental Figure S1. Expression levels of defense-related genes in the Col-0
plants of A. thaliana inoculated with Y1H2Y3.
Supplemental Figure S2. In situ association between Y2b and CAT3 in Arabidopsis
23
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Copyright © 2011 American Society of Plant Biologists. All rights reserved.
cells.
Supplemental Figure S3. Characterization of the Col-CAT transgenic plants.
ACKNOWLEDGMENTS
We thank Kae Sueda, Eri Yoshimoto and Shunichi Tanaka for their technical
assistance.
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FIGURE LEGENDS
29
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Copyright © 2011 American Society of Plant Biologists. All rights reserved.
Figure 1. Necrosis induction and H2O2 generation by Y1H2Y3, a pseudorecombinant
between Cucumber mosaic virus-Y (CMV-Y) and CMV-HL, in plants of Arabidopsis
thaliana Col-0 ecotype. A, Necrotic symptoms on the plants infected with CMV-HL and
Y1H2Y3 at 18 days post-inoculation (dpi). B, Symptoms on CMV-infected plants at 10
dpi. C, Detection of H2O2 in the Col-0 plants infected with Y1H2Y3 using the
H2O2-sensitive fluorescent probe H2DCFDA. Leaves were harvested at 10 dpi and
photographed using either bright-field light microscopy (BF) or by fluorescent
microscopy (H2O2). Note that necrosis was associated with the sites that fluoresced. D,
Symptomatic Col-0 leaves (upper panels) were subjected to tissue printing (lower
panels) to localize CMV.
Figure 2. Interaction between HL2b and catalase in the yeast two-hybrid assay. Each
plasmid (pGADT7, pGADT7-CAT1, pGADT7-CAT2 and pGADT7-CAT3) was
co-transferred with pGBKT7-HL2b into Yeast strain AH109. The transformed AH109
cells were plated on the growth medium without leucine, tryptophan (-LW, left panel),
or without leucine, tryptophan, histidine and adenine (-LWHA, right panel). Only when
the expressed proteins bind to each other, the cells can grow on the synthetic medium.
Figure 3. CMV-HL 2b interacts with Arabidopsis Catalase 3 (CAT3). A, Schematic
representation of the C-terminal deletion mutants of HL2b for the yeast two-hybrid
assay. Deleted forms of the HL2b gene were fused to the binding domain in the
pGBKT7 vector (Clontech) and the CAT3 ORF was inserted in pGADT7 activation
30
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Copyright © 2011 American Society of Plant Biologists. All rights reserved.
domain vector (Clontech). Transcriptional activity of β-galactosidase was assayed. Plus
(+) indicates transcriptional activation while minus (-) indicates a negative result. B,
Symptoms in the Col-0 plants infected with C2-H1-HL2b or C2-H1-HL2bΔ40. The
recombinant HL2bs (HL2b, HL2bΔ12, HL2bΔ33 and HL2bΔ40) were inserted in the
CMV vector, C2-H1 for virus inoculation experiments. No necrotic symptom was
observed in the Col-0 plants infected with C2-H1-HL2bΔ40. C, CMV accumulation
levels. ELISA was conducted to analyze the CMV accumulation in Col-0 plants. Error
bars represent standard errors of the means.
Figure 4. Catalase activity, transcripts and protein in the CMV-infected Col-0 plants. A,
Catalase activity in CMV-Y-, mock- and Y1H2Y3-inocualted plants was determined by
measuring the decrease in absorbance at 240 nm 10 dpi. Catalase activity was expressed
as mmol⋅−1min⋅−1mg protein. Three replicates were done. Error bars represent standard
errors of the means. C2-H1 lacks the entire 2b. B, The CAT (CAT1, CAT2 and CAT3)
expression levels in the Col-0 plants inoculated with Y1H2Y3 were determined by
quantitative RT-PCR 2 weeks after mock and CMV inoculation. Error bars represent
standard error of the mean of three replicates. C, Western blot analysis was performed
using the specific anti-CAT3 antibody. Total protein was extracted from the
CMV-inoculated leaves at 4 dpi and from the upper leaves at 4 and 7 dpi. Note that
there was little difference in the expression level of CAT3 between Mock- and
CMV-inoculated plants, indicating that CMV does not affect CAT3 accumulation.
31
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Copyright © 2011 American Society of Plant Biologists. All rights reserved.
Figure 5. Necrosis induction and H2O2 generation in transgenic Col-0 plants expressing
HL2b. A, Necrotic symptoms in the leaves of transgenic plants expressing HL2b
(Col-HL2b). WT, Col-0. B, Western blot analysis of the 2b protein in the transgenic
plants. Total proteins (30 μg), which were extracted from Col-0 (WT), CMV-infected
leaves (Y1H2Y3) and Col-HL2b (lines 1, 6 and 7), were separated by SDS-PAGE and
blotted onto a PVDF membrane. The blots were then subjected to immunodetection
using anti-2b antibodies. C, Detection of H2O2 in the Col-HL2b lines using the
H2O2-sensitive fluorescent probe H2DCFDA. Note that necrosis on the edge of the
leaves that were harvested from the transgenic plants was associated with fluorescent
spots. D, Catalase activities in the Col-HL2b lines. Catalase activity was determined as
in the legend of Figure 4.
Figure 6. In situ association between HL2b and CAT3 in Arabidopsis cells. Protoplasts
were transfected with HL2b, CAT3-FLAG or HL2b+CAT3-FLAG. BF indicates the
image observed with bright-field light microscopy. Protoplast nuclei were visualized
using Hoechst 33342 staining and UV light (UV). For detection of in situ molecular
interaction, the Duolink in situ PLA kit was used. A, Interaction between HL2b and
CAT3. Two primary antibodies against FLAG and CMV 2b were used to detect CAT3
and 2b, respectively. With fluorescent microscopy, PLA-red signals from Texas red
(TX2) will theoretically be detected only when there is an in vivo interaction between
2b and CAT3. The images of UV and TX2 were merged (merge column) to identify the
exact localization of the signals. B, Subcellular localization of CAT3-FLAG in
32
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Copyright © 2011 American Society of Plant Biologists. All rights reserved.
protoplasts. CAT3 was detected using anti-FLAG antibodies as red signals of TX2. Note
that CAT3 was detected in nuclei when HL2b was expressed with the CAT3 gene, while
the protein was dispersed in cells without HL2b. C, Subcellular localization of HL2b in
protoplasts. HL2b was detected using anti-2b antibodies, which yielded PLA-red signals
from TX2. As expected, HL2b was localized in nuclei.
Figure 7. Characterization of Col-CAT transgenic (lines 4, 6 and 7) plants inoculated
with Y1H2Y3. A, Delay of necrosis in Col-CAT transgenic plants compared to the wild
type Col-0 (WT) at 10 dpi. B, Detection of H2O2 in control and Col-CAT plants
inoculated with Y1H2Y3 using H2DCFDA. Leaves were harvested at 10 dpi and
photographed either by light microscopy (BF) or by fluorescent microscopy (H2O2). C,
Development of necrotic symptoms in control and Col-CAT transgenic (lines 4, 6 and 7)
plants. Percentage of symptomatic plants was calculated using 10 plants for each
transgenic line.
Figure 8. Effect of the interaction between CAT3 and HL2b on cell death of
Arabidopsis Col-0 protoplasts. A, Percentage of cell death for transfected Col-0
protoplasts at 20 h after transfection with GFP (control, 10 μg), HL2b (5 μg)+GFP (5
μg), GFP (5 μg)+CAT3 (5 μg) or HL2b (5 μg)+CAT3 (5 μg). Cells that were stained by
Evans blue were scored as dead. Data are percentage of cell death after the control
percentage was subtracted from the background. Error bars represent standard errors of
the means of three replicates. Differences in the means were evaluated with a t test; **
33
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Copyright © 2011 American Society of Plant Biologists. All rights reserved.
significant at 1% level, respectively. B, Catalase activities in Col-0 protoplasts
transfected with GFP (control), HL2b+GFP, GFP+CAT3 or HL2b+CAT3 at 20 h after
transfection. Activity was measured spectrophotometrically. Error bars represent
standard errors of the means of three replicates. Differences in means among groups
were evaluated by t test; * significant at 5% level.
Figure 9. Enhanced resistance to CMV by CAT3 overexpression in protoplasts and
Col-CAT transgenic plants. A, Effect of CAT3 expression on the accumulation of
Y1H2Y3 in Col-CAT plants. Quantitative RT-PCR was performed to measure the
accumulation of CMV in Col-CAT transgenic plants inoculated with Y1H2Y3. The
values were normalized using the mRNA levels of the β-tublin gene. The value for the
non-transgenic Col-0 plant at 4 dpi was set at 1.0. The values represent means with
standard errors obtained from three replicates. Differences in means among groups were
evaluated by t test; * significant at 5% level. B, Effect of CAT3 overexpression on the
RNA silencing suppressor activity of HL2b. N. benthamiana protoplasts were
co-transfected with pBI-Fluc (0.3 µg), pE-Rluc (0.03 µg), dsFluc (0.03 µg) and viral
suppressor (HL2b or P19, 3 μg of plasmid) together with CAT3 (0, 1 and 3 μg of
plasmid). GFP (3 μg) was used as a control. At 20 h after transfection, Fluc activities in
the treated protoplasts were measured. Fluc activity was then divided by Rluc activity,
and the value for the mock was set at 1.0. The values represent the means with standard
deviations (SD) from three replicates. Note that addition of CAT3 decreased the RSS
activity of HL2b, but did not affect that of P19.
34
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Figure 10. Cellular localization of CAT3 and cell death induction in protoplasts
transfected with HL2bΔNLS. A, Schematic representation of HL2bΔNLS. The mutants
had deletions of the two nuclear localization signals (NLS1 and NLS2). B, In situ
association between HL2bΔNLS and CAT3 in Arabidopsis cells. Protoplasts isolated
from Col-0 plants were transfected with HL2bΔNLS+CAT3, and harvested after 20 h.
The TX2 signals are theoretically detected only when 2b and CAT3 interact in vivo. C,
Subcellular localization of CAT3-FLAG in protoplasts. CAT3 was detected using
anti-FLAG antibodies as red signals of TX2. D, Subcellular localization of HL2bΔNLS
in protoplasts. HL2bΔNLS was detected using anti-CMV 2b antibodies as red signals of
TX2. E, Cell death of Col-0 protoplasts transfected with HL2bΔNLS. The protoplasts
were transfected with GFP (control), HL2b or HL2bΔNLS. At 20 h after transfection,
cell death was scored by staining the protoplasts with Evans blue. Data are presented as
a percentage of cell death. Three replicates were done. Error bars represent standard
error of the means.
35
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Copyright © 2011 American Society of Plant Biologists. All rights reserved.
A
Mock CMV-Y Y1H2Y3 CMV-HL
B
Mock
CMV-Y
Y1H2Y3
D
C
Mock CMV-Y Y1H2Y3
Mock
CMV-Y Y1H2Y3
BF
H 2O 2
Figure 1. Necrosis induction and H2O2 generation by Y1H2Y3, a
pseudorecombinant between Cucumber mosaic virus-Y (CMV-Y) and CMVHL, in plants of Arabidopsis thaliana Col-0 ecotype. A, Necrotic symptoms
on the plants infected with CMV-HL and Y1H2Y3 at 18 days postinoculation (dpi). B, Symptoms on CMV-infected plants at 10 dpi. C,
Detection of H2O2 in the Col-0 plants infected with Y1H2Y3 using the H2O2sensitive fluorescent probe H2DCFDA. Leaves were harvested at 10 dpi and
photographed using either bright-field light microscopy (BF) or by
fluorescent microscopy (H2O2). Note that necrosis was associated with the
sites that fluoresced. D, Symptomatic Col-0 leaves (upper panels) were
subjected to tissue printing (lower panels) to localize CMV.
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Copyright © 2011 American Society of Plant Biologists. All rights reserved.
-LW
-LWHA
pGBKT7-HL2b
+
pGADT7
pGBKT7-HL2b
+
pGADT7-CAT1
pGBKT7-HL2b
+
pGADT7-CAT2
pGBKT7-HL2b
+
pGADT7-CAT3
Figure 2. Interaction between HL2b and catalase in the yeast two-hybrid
assay. Each plasmid (pGADT7, pGADT7-CAT1, pGADT7-CAT2 and
pGADT7-CAT3) was co-transferred with pGBKT7-HL2b into Yeast strain
AH109. The transformed AH109 cells were plated on the growth medium
without leucine, tryptophan (-LW, left panel), or without leucine, tryptophan,
histidine and adenine (-LWHA, right panel). Only when the expressed
proteins bind to each other, the cells can grow on the synthetic medium.
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Copyright © 2011 American Society of Plant Biologists. All rights reserved.
A
(aa)
2b
HL2b
HL2bΔ12
HL2bΔ33
HL2bΔ40
C
C2-H1- C2-H1HL2b HL2bΔ40
Necrosis
+
+
+
+
+
+
-
-
1
0.8
OD405
B
Yeast
-galactosidase
assay
0.6
0.4
0.2
0
C2-H1-HL2b C2-H1-HL2bΔ40
Figure 3. CMV-HL 2b interacts with Arabidopsis Catalase 3 (CAT3). A,
Schematic representation of the C-terminal deletion mutants of HL2b for the
yeast two-hybrid assay. Deleted forms of the HL2b gene were fused to the
binding domain in the pGBKT7 vector (Clontech) and the CAT3 ORF was
inserted in pGADT7 activation domain vector (Clontech). Transcriptional
activity of -galactosidase was assayed. Plus (+) indicates transcriptional
activation while minus (-) indicates a negative result. B, Symptoms in the Col0 plants infected with C2-H1-HL2b or C2-H1-HL2bΔ40. The recombinant
HL2bs (HL2b, HL2bΔ12, HL2bΔ33 and HL2bΔ40) were inserted in the
CMV vector, C2-H1 for virus inoculation experiments. No necrotic symptom
was observed in the Col-0 plants infected with C2-H1-HL2bΔ40. C, CMV
accumulation levels. ELISA was conducted to analyze the CMV accumulation
in Col-0 plants. Error bars represent standard errors of the means.
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A
Catalase activity
1.2
1
0.8
0.6
0.4
0.2
0
B
Relative expression levels
Mock CMV-Y
C2-H1 Y1H2Y3
1.2
1
0.8
Mock
0.6
CMV-Y
0.4
Y1H2Y3
0.2
0
CAT1
CAT2
CAT3
C
Inoculated leaf
4 days
Upper leaf
4 days
7 days
Figure 4. Catalase activity, transcripts and protein in the CMV-infected Col-0 plants.
A, Catalase activity in CMV-Y-, mock- and Y1H2Y3-inocualted plants was
determined by measuring the decrease in absorbance at 240 nm 10 dpi. Catalase
activity was expressed as mmol1min1mg protein. Three replicates were done. Error
bars represent standard errors of the means. C2-H1 lacks the entire 2b. B, The CAT
(CAT1, CAT2 and CAT3) expression levels in the Col-0 plants inoculated with
Y1H2Y3 were determined by quantitative RT-PCR 2 weeks after mock and CMV
inoculation. Error bars represent standard error of the mean of three replicates. C,
Western blot analysis was performed using the specific anti-CAT3 antibody. Total
protein was extracted from the CMV-inoculated leaves at 4 dpi and from the upper
leaves at 4 and 7 dpi. Note that there was little difference in the expression level of
CAT3 between Mock- and CMV-inoculated plants, indicating that CMV does not
affect CAT3 accumulation.
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A
WT
Col-HL2b
line6
line1
B
line1
line7
Col-HL2b
line6
line7
HL2b
C
WT
Col-HL2b
line1
line6
line7
BF
H2 O2
D
Catalase activity
1.2
1
0.8
0.6
0.4
0.2
0
WT
line1
line6
line7
Col-HL2b
Figure 5. Necrosis induction and H2O2 generation in transgenic Col-0 plants
expressing HL2b. A, Necrotic symptoms in the leaves of transgenic plants
expressing HL2b (Col-HL2b). WT, Col-0. B, Western blot analysis of the HL2b
protein in the transgenic plants. Total proteins (30 g), which were extracted
from Col-0 (WT), CMV-infected leaves (Y1H2Y3) and Col-HL2b (lines 1, 6
and 7), were separated by SDS-PAGE and blotted onto a PVDF membrane. The
blots were then subjected to immunodetection using anti-2b antibodies. C,
Detection of H2O2 in the Col-HL2b lines using the H2O2-sensitive fluorescent
probe H2DCFDA. Note that necrosis on the edge of the leaves that were
harvested from the transgenic plants was associated with fluorescent spots. D,
Catalase activities in the Col-HL2b lines. Catalase activity was determined as in
the legend of Figure. 4.
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A Interaction between HL2b and CAT3
B
C
BF
Mock
UV TX2 merge
BF
CAT3-FLAG
UV TX2 merge
BF
HL2b
UV TX2 merge
HL2b + CAT3-FLAG
BF UV TX2 merge
Subcellular localization of CAT3-FLAG
CAT3-FLAG
HL2b
BF UV TX2 merge
BF UV TX2 merge
HL2b + CAT3-FLAG
BF UV TX2 merge
Subcellular localization of HL2b
HL2b
CAT3-FLAG
BF UV TX2 merge
BF UV TX2 merge
HL2b + CAT3-FLAG
BF UV TX2 merge
Figure 6. In situ association between HL2b and CAT3 in Arabidopsis cells. Protoplasts
were transfected with HL2b, CAT3-FLAG or HL2b+CAT3-FLAG. BF indicates the
image observed with bright-field light microscopy. Protoplast nuclei were visualized using
Hoechst 33342 staining and UV light (UV). For detection of in situ molecular interaction,
the Duolink in situ PLA kit was used. A, Interaction between HL2b and CAT3. Two
primary antibodies against FLAG and CMV 2b were used to detect CAT3 and 2b,
respectively. With fluorescent microscopy, PLA-red signals from Texas red (TX2) will
theoretically be detected only when there is an in vivo interaction between 2b and CAT3.
The images of UV and TX2 were merged (merge column) to identify the exact localization
of the signals. B, Subcellular localization of CAT3-FLAG in protoplasts. CAT3 was
detected using anti-FLAG antibodies as red signals of TX2. Note that CAT3 was detected
in nuclei when HL2b was expressed with the CAT3 gene, while the protein was dispersed
in cells without HL2b. C, Subcellular localization of HL2b in protoplasts. HL2b was
detected using anti-2b antibodies, which yielded PLA-red signals from TX2. As expected,
HL2b was localized in nuclei.
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A
Y1H2Y3infected
WT
B
Y1H2Y3infected Col-CAT
line6
line7
line4
Y1H2Y3Y1H2Y3infected Col-CAT
infected
WT
line4
line6
line7
BF
H2O2
C
Percentages of the plants
showing necrosis
100
%
80
Col-0 WT
60
Col-CAT line4
Col-CAT line6
40
Col-CAT line7
20
0
6
7
8
9
10
11
12
13
14
15
16
17
Days after Y1H2Y3 inoculation
Figure 7. Characterization of Col-CAT transgenic (lines 4, 6 and 7) plants
inoculated with Y1H2Y3. A, Delay of necrosis in Col-CAT transgenic plants
compared to the wild type Col-0 (WT) at 10 dpi. B, Detection of H2O2 in control
and Col-CAT plants inoculated with Y1H2Y3 using H2DCFDA. Leaves were
harvested at 10 dpi and photographed either by light microscopy (BF) or by
fluorescent microscopy (H2O2). C, Development of necrotic symptoms in control
and Col-CAT transgenic (lines 4, 6 and 7) plants. Percentage of symptomatic
plants was calculated using 10 plants for each transgenic line.
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Percentage of cell death
A
B
25
**
20
15
10
5
0
GFP
HL2b
+
GFP
GFP
+
CAT3
1.4
*
1.2
Catalse activity
HL2b
+
CAT3
1
0.8
0.6
0.4
0.2
0
GFP
HL2b
+
GFP
GFP
+
CAT3
HL2b
+
CAT3
Figure 8. Effect of the interaction between CAT3 and HL2b on cell death of
Arabidopsis Col-0 protoplasts. A, Percentage of cell death for transfected
Col-0 protoplasts at 20 h after transfection with GFP (control, 10 μg), HL2b
(5 μ g)+GFP (5 μg), GFP (5 μg)+CAT3 (5 μg) or HL2b (5 μg)+CAT3 (5 μg).
Cells that were stained by Evans blue were scored as dead. Data are
percentage of cell death after the control percentage was subtracted from the
background. Error bars represent standard errors of the means of three
replicates. Differences in the means were evaluated with a t test; **
significant at 1% level, respectively. B, Catalase activities in Col-0
protoplasts transfected with GFP (control), HL2b+GFP, GFP+CAT3 or
HL2b+CAT3 at 20 h after transfection. Activity was measured
spectrophotometrically. Error bars represent standard errors of the means of
three replicates. Differences in means among groups were evaluated by t test;
* significant at 5% level.
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A
Relative RNA levels
CMV / β-tubulin
3
*
**
2.5
Col-0
2
Col-CAT line4
1.5
Col-CAT line6
1
Col-CAT line7
0.5
0
4
7
14
B
Fluc / Rluc
1.5
1.0
0.5
0
Mock
GFP
HL2b HL2b
+
CAT3
(1 g)
Mock
GFP
P19
HL2b
+
CAT3
(3 g)
Fluc / Rluc
1.5
1.0
0.5
0
P19
P19
+
+
CAT3 CAT3
(1 g) (3 g)
Figure 9. Enhanced resistance to CMV by CAT3 overexpression in
protoplasts and Col-CAT transgenic plants. A, Effect of CAT3 expression on
the accumulation of Y1H2Y3 in Col-CAT plants. Quantitative RT-PCR was
performed to measure the accumulation of CMV in Col-CAT transgenic
plants inoculated with Y1H2Y3. The values were normalized using the
mRNA levels of the -tublin gene. The value for the non-transgenic Col-0
plant at 4 dpi was set at 1.0. The values represent means with standard errors
obtained from three replicates. Differences in means among groups were
evaluated by t test; * significant at 5% level. B, Effect of CAT3
overexpression on the RNA silencing suppressor activity of HL2b. N.
benthamiana protoplasts were co-transfected with pBI-Fluc (0.3 µg), pERluc (0.03 µg), dsFluc (0.03 µg) and viral suppressor (HL2b or P19, 3 g of
plasmid) together with CAT3 (0, 1 and 3 g of plasmid). GFP (3 g) was
used as a control. At 20 h after transfection, Fluc activities in the treated
protoplasts were measured. Fluc activity was then divided by Rluc activity,
and the value for the mock was set at 1.0. The values represent the means
with standard deviations (SD) from three replicates. Note that addition of
CAT3 decreased the RSS activity of HL2b, but did not affect that of P19.
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A
B
Interaction between
HL2bΔNLS and CAT3
HL2bΔNLS+CAT3-FLAG
BF UV TX2 merge
HL2bΔNLS
(aa)
NLS1 NLS2
C
D
Subcellular localization of CAT3-FLAG
Subcellular localization of HL2bΔNLS
HL2bΔNLS+CAT3-FLAG
BF UV TX2 merge
HL2bΔNLS+CAT3-FLAG
BF UV TX merge
2
E
Percentage of
cell death
40
30
20
10
0
GFP
HL2b HL2bΔNLS
Figure 10. Cellular localization of CAT3 and cell death induction in
protoplasts transfected with HL2bΔNLS. A, Schematic representation of
HL2bΔNLS. The mutants had deletions of the two nuclear localization
signals (NLS1 and NLS2). B, In situ association between HL2bΔNLS and
CAT3 in Arabidopsis cells. Protoplasts isolated from Col-0 plants were
transfected with HL2bΔNLS+CAT3-FLAG, and harvested after 20 h. The
TX2 signals are theoretically detected only when 2b and CAT3 interact in
vivo. C, Subcellular localization of CAT3-FLAG in protoplasts. CAT3 was
detected using anti-FLAG antibodies as red signals of TX2. D, Subcellular
localization of HL2bΔNLS in protoplasts. HL2bΔNLS was detected using
anti-CMV 2b antibodies as red signals of TX2. E, Cell death of Col-0
protoplasts transfected with HL2bΔNLS. The protoplasts were transfected
with GFP (control), HL2b or HL2bΔNLS. At 20 h after transfection, cell
death was scored by staining the protoplasts with Evans blue. Data are
presented as a percentage of cell death. Three replicates were done. Error
bars represent standard error of the means.
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